Adult cardiac valve disease affects up to 5% of Americans with approximately 25% of the aged population developing aortic valve sclerosis, a marker of valve disease. The primary treatment of valvular insufficiency is surgical intervention with an estimated cost of $1 billion per year in the US. The fact that most diseased valves have an underlying malformation suggests that determining the etiology of valve diseases lies in the discovery of novel mechanisms and genes that are involved in valve development. However, progress in discovering the roots of valve disease has been limited in part due to the lack of viable mouse models that exhibit significant penetrance of a cardiac valve phenotype. A hallmark of both developmental myxomatous valve abnormalities and adult disease is the loss of stratification of extracellular matrix (ECM) and the accumulation of the ECM proteoglycans including versican. The ADAMTS (ADisintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) proteoglycanases are a specific class of extracellular matrix metalloproteases that cleave proteoglycans. Mice deficient in ADAMTS5 develop severely enlarged cardiac valves by late gestation with significant accumulation of versican, the ECM substrate of ADAMTS5. In vivo reduction of versican, rescues the Adamts5-/- valve phenotype, and demonstrates that cleavage of versican is required for its clearance from the provisional ECM. To date, versican is the only ECM substrate of ADAMTS5 known to accumulate in ADAMTS5 deficient valves. Prevalvular mesenchymal cells from the Adamts5-/- mice maintain Sox9 expression and show elevated levels of proliferation suggesting they do not differentiate like their WT counterparts. Our published data also show that ECM accumulation of versican inhibits Smad2 phosphorylation. Intergenetic cross of the Adamts5-/- with Smad2-/- exacerbates the valve phenotype and results in a high penetrance (80%) of bicuspid aortic and pulmonary valves. In this application we seek to elucidate ECM-cell signaling mechanisms that are activated in response to proteoglycan cleavage and integral to the valve maturation and homeostasis. Since ADAMTS5 peptide inhibitors are in clinical trials to alleviate osteoarthritis, investigation into the requirement of proteoglycan cleavage in normal valve development and homeostasis has immediate clinical relevance. The three specific aims of our application test our overarching hypothesis that ADAMTS5 mediated versican cleavage plays a pivotal role in semilunar cardiac valve maturation through interconnections with TGFbeta signaling that control cell differentiation and the balance of proteoglycan-rich and fibrous ECM. Aim 1: Determine the mechanism by which ADAMTS5 dependent ECM remodeling upregulates Smad2 phosphorylation during semilunar valve development. Aim 2: Determine the spatiotemporal expression of ADAMTS5 that is required to remodel the provisional ECM of endocardial cushions during semilunar valve development. Aim 3: Determine the consequence of ADAMTS5 deficiency on adult murine valve ECM and function.